Under various cell environments, most biological cells can drastically change their shape (called cell "deformability") without structural damage to the cell membrane, without a loss in cell contents to the cell environment, and without a change in the chemical function of the cell membrane. Under other cell environments, the cell membrane can lose chemical function, can suffer structural damage, and can rupture to lose cell contents to the cell environment (called cell fragility). Thus, cell deformability and cell fragility are two independent characteristics of a cell membrane. Cells can have any combination of a high-to-low deformability and a high-to-low fragility. However, most normal cells have high deformability and moderate-to-low fragility.
Deformability is one important characteristic for some cells such as "sensory receptors" which are normally stationary in the cell environment and for other cells such as blood and lymph cells which normally move in the cell environment. Cell fragility is an important characteristic for almost all normally stationary and normally moving cells because changes in the cell environment can cause considerable amounts of water to move into cells to rupture more fragile cells. Changes in cell fragility can change the ability of a cell to perform its normal function in a normal or abnormal cell environment. For example, an abnormally high fragility of red blood cells will lead to premature rupture of many red blood cells in a normal blood environment which will reduce the circulating pool of red blood cells and will reduce the red blood cell transport of oxygen to tissues. Yet, these highly fragile cells can have normal cell deformability. Thus, fragility might clinically be a more important cell characteristic than deformability.
Cell fragility is altered by many conditions such as cell age, duration of blood-bank storage, treatment with a variety of membrane-binding drugs, and progression of membrane or hemoglobin-related diseases such as diabetes and sickle-cell anemia, respectively. Thus, a rapid, highly accurate, and easily applied method is needed for clinical measurements to assess cell fragility as an index of "cell strength" which is defined as the degree to which the cell membrane can maintain its structural integrity and its chemical function in a normal and an altered cell environment. Most current methods for assessing cell strength primarily create mechanical forces on cells to assess either the deformability or the fragility of these cells. These methods include Osmotic-Gradient Ektacytometry, and Cell Filtration, Micropipette Suction, and Osmotic Fragility tests.
Osmotic-Gradient Ektacytometry is one method that applies mechanical forces to measure a combination of cell deformability and cell fragility. This method uses a viscometer device to measure shape changes which are induced in red blood cells by various speeds of rotation (called applied shear stress) of the test cells with different osmotic solutions in the cell environment. The osmotic-spectrum curves which are produced by Ektacytometry are highly variable for the same test-cell sample due to small changes in sample ambient temperature, pH, and plasma osmolality. Thus, these osmotic-spectrum curves are complex and very difficult to use for interpretation of changes in cell deformability. As a result, Ektacytometry currently requires very sophisticated equipment, extensive operator training, and highly controlled test conditions to obtain one measurement value for the combination of cell deformability and fragility. This makes Ektacytometry usable only in a few research laboratories and not in a clinical setting.
Other methods such as Cell Filtration and Micropipette Suction also require the external application of a mechanical force to cells but these methods primarily measure cell deformability by forcing (filtering) cells through various size pores or by mechanical aspiration of these cells into micropipettes of fixed tip size and taper. These methods also require very sophisticated equipment and substantial operator training; and they are extremely time consuming to obtain an analysis of only a relatively few cells in each test-cell sample. Thus, these methods have also not been accepted into general clinical use.
Assessment of cell deformability in the Cell Filtration method has been improved by measurements of electrical impedance (Hanss et al, U.S. Pat. No. 4,835,457) and measurements of time (David D. Paterson, U.S. Pat. No. 4,491,012) during the application of external mechanical force (pressure) to force red blood cells to pass through either an artificial membrane filter or a foil system (Helmut Jahn, U.S. Pat. No. 4,797,606). These measurement methods are extremely sensitive to manufacturing tolerances on the filter or foil and both of these measurement methods primarily assess only cell deformability and not cell fragility. Thus, these measurement additions to the Cell Filtration method have also not found their way into common clinical use.
The Osmotic Fragility method was one of the earliest techniques that was developed for assessment primarily of red blood cell fragility rather than deformability, and it is one of the few methods currently in clinical use. The Osmotic Fragility method is time-consuming, and it requires multiple blood handling steps, and relatively large volumes of blood samples. The Osmotic Fragility method uses exposure of blood samples to a large range of salt concentrations to produce a large range in the osmotic pressure for the cell environment. The osmotic pressure mechanically forces water into cells to swell cells to the point of cell membrane rupture.
The osmotic pressure method has been adapted by Groves and Rodriguez (U.S. Pat. No. 4,535,284) to apply high frequency and low frequency electrical currents to detect the percent of red blood cells that are altered during application of osmotic mechanical forces over a large range of osmotic cell environments. This use of high frequency and low frequency electrical currents is combined with a Coulter Counter.RTM. to classify individual red blood cells as normal or abnormal.
Overall, the osmotic mechanical gradient method and various refinements to this method can only detect relatively large changes in cell membrane fragility because this osmotic-based mechanical method provides no information about the rate of cell lysis (which occurs when the cell membrane ruptures). The lack of method sensitivity to mild or moderate changes in cell fragility has led to the clinical use of the osmotic mechanical method only for diagnosis of one disease called hereditary spherocytosis.
In contrast to methods that apply external mechanical forces mostly to measure cell deformability or in one case (osmotic gradients) to measure cell fragility, there is a chemical method which changes the mechanical characteristics of cell membranes to assess cell membrane fragility. It was first shown some fifty years ago that some chemicals can be changed by light (called photoactivation) to induce the rupture or breakup of red blood cell membranes (called hemolysis) in a test tube. Since then, this basic process (called photohemolysis) has been extensively studied in a variety of test-tube experiments.
The mechanism for photohemolysis is oxygen dependent and is thought to involve the generation of singlet oxygen with the subsequent oxidation of proteins in the red blood cell membrane. It has been suggested that this protein oxidation leads to the creation of extra water channels in the cell membrane. This would increase passive cationic exchange across the cell membrane to give a subsequent influx of water into the cell to swell the cell to the point of hemolysis. It has been suggested that photohemolysis could also involve peroxidation of the lipid layers in the cell membrane. This peroxidation appears to limit the ability of molecules to move (called membrane fluidity) in the lipid bilayer of the cell membrane, which then appears to limit the ability of the cell to undergo shape changes (cell deformability), since shape changes in normal cells appear to depend on membrane fluidity in the lipid bilayer.
Thus, there is considerable scientific evidence to show that certain chemical agents can be photoactivated to create cell-attack agents which disrupt red blood cells by altering either the protein or the lipid layer of the cell membrane. These alterations in cell membrane structure permit water inflow to increase internal cell volume until the "internal cell pressure" ruptures the cell membrane (called cell fragility) sufficiently to cause the loss of cell contents (called lysis in general or hemolysis in the case of red blood cells).
Currently, a limited number of chemical agents have been identified as potential cell-attack agents. Previous research has not yet discovered an acceptable method for use of these cell-attack agents to reproducibly create photohemolysis. Previous uses of these cell-attack agents have been limited by requirements for long photoactivation periods, long analysis times, test-cell isolation from the original blood sample, multiple blood samples and high volume (milliliter) samples, and by a test result that gives only a single measurement parameter for each sample.
The long photoactivation periods have been a major limitation. For example, photoactivation of red blood cells incubated with 0.1 mM of protoporphyrin as the cell-attack agent requires approximately twenty minutes of photoactivation exposure time to achieve only a modest 20% hemolysis in the cell sample; a 100% hemolysis requires a 25-minute or longer photoactivation exposure period with this agent. Similarly, more than twenty minutes of photoactivation exposure is needed with pheophorbide as the cell-attack agent to give approximately 90% hemolysis in the cell sample. Photoactivation periods of 3-4 hours are required with eosin-isothiocyanate as the cell-attack agent to give maximal hemolysis which only then occurs approximately 11 hours after the photoactivation period. These long photoactivation periods produce highly variable hemolysis rates and hemolysis levels which have limited these photohemolysis techniques to research use.
A few researchers have previously attempted the use of a light scattering device to detect the presence of significant photohemolysis. However, this device is very sensitive to very small changes in red blood cell concentrations, and this device requires a monolayer of red blood cells with a red cell concentration of less than 0.00025%. Such concentrations have not been practicable to achieve even by current modern micropipetting systems. In addition, the use of a cell monolayer and the light scattering measurement have also been limited by a long photoactivation and hemolysis measurement period since 100% hemolysis of a cell sample requires four hours with phloxine B as the photoactivated cell-attack agent.
Current clinical methods rely on osmotic mechanical forces over a wide osmotic range to create hemolysis, and these methods require milliliter (i.e. macro) quantities of blood volumes to determine an osmolality range (equivalent to internal water pressure) within which hemolysis occurs. These Osmotic Fragility tests are generally performed on sample volumes in test tubes or cuvettes, and parallel light is required for detection of the presence or absence of significant hemolysis. These current clinical hemolysis methods cannot separately measure changes in cell deformability and cell fragility, cannot distinguish changes in cell membrane fragility due to alterations in the protein layer of the membrane from fragility changes due to alterations in the lipid layers of the membrane, and cannot determine "a rate of hemolysis" which would provide a more sensitive hemolysis test for clinical use.
While the micro-analysis process of the instant invention often employs a method and test conditions which control the osmolality of the micro-sample, no particular osmolality is necessary for the micro-lysis and analysis process and it is not osmolality dependent. Controlling the osmolality of the micro-sample is merely a convenience.
Both the current Osmotic Fragility clinical method and the Photohemolysis research method require multiple cell samples, multiple sample dilutions, sample centrifugation, and separate sample analysis in a spectrophotometer to give a single measurement of a parameter called Percent Cell Hemolysis at one point in time as an assessment of cell membrane fragility. Thus, these current clinical and research methods do not provide simultaneous energy-dose dependent and time-dependent measurements of photoactivation and hemolysis relationships from one, small volume (i.e. micro), blood sample.